Enhanced transgene expression and processing
Inventors
Le Fourn, Valerie • Mermod, Nicolas • Regamey, Alexandre • Buceta, Montse • Ley, Deborah • Harraghy, Niamh • Kostyrko, Kaja • Giro, Pierre-Alain • Calabrese, David
Assignees
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Publication Number
US-11898154-B2
Publication Date
2024-02-13
Expiration Date
Abstract
Disclosed are constructs and methods for expressing DNAs of interest in particular in non-primate eukaryotic host cells that display advantages with regard quantity and quality of expression including high stability of expression and, if appropriate, transport of the expression product out of the cell.
Core Innovation
The invention provides a recombinant nucleic acid molecule that forms part of a transposon vector comprising a 5′ transposon-specific inverted terminal repeat (ITR) and a 3′ ITR, with a region located between the 5′ ITR and the 3′ ITR. This region includes at least one nucleic acid sequence encoding a transgene expression processing (TEP) protein under the control of a promoter. The TEP is selected from hSRP14, hSec61α, hSec61β, hSec61γ, hSRP54, hSRP9, hSRPRα, hSRPβ, hCANX, or combinations thereof.
The recombinant nucleic acid molecule further includes a matrix attachment region (MAR) element. In combination with the transposon vector, the MAR element and the TEP protein are stated to increase expression of a transgene encoding a product of interest in a mammalian cell by at least 10% relative to a plasmid vector comprising the TEP protein.
The document also describes MAR architectures including singular MAR elements and engineered/rearranged MAR derivatives. Dependent content further constrains MAR sequence identity to defined SEQ ID numbers and constrains acceptable TEP protein sequences using amino acid sequence identity thresholds.
Claims Coverage
The independent claim identified is clm-00001. It contains a set of inventive features centered on a transposon vector architecture that combines 5′/3′ ITRs, a TEP protein expression cassette under a promoter, and a MAR element to increase mammalian transgene expression versus a plasmid vector.
Transposon vector with 5′ and 3′ ITRs and an intervening expression region
A recombinant nucleic acid molecule that is part of a transposon vector comprising a 5′ transposon-specific inverted terminal repeat (ITR) and a 3′ ITR, and a region located between the 5′ ITR and the 3′ ITR.
TEP protein cassette under promoter control within the transposon region
At least one nucleic acid sequence encoding a transgene expression processing (TEP) protein under the control of a promoter, wherein the TEP is selected from the group consisting of human Signal Recognition Particle 14 (hSRP14), human Protein Transport Protein 61α (hSec61α), human Protein Transport Protein 61β (hSec61β), human Protein Transport Protein 61γ (hSec61γ), human Signal Recognition Particle 54 (hSRP54), human Signal Recognition Particle 9 (hSRP9), human Signal Recognition Particle Receptor α (hSRPRα), human Signal Recognition Particle β (hSRPβ), human Calnexin (hCANX), and combinations thereof.
MAR element included between the 5′ and 3′ ITRs
A matrix attachment region (MAR) element located between the 5′ ITR and the 3′ ITR.
Improved mammalian transgene expression versus TEP plasmid vector
The transposon vector increases an expression of a transgene encoding a product of interest in a mammalian cell by at least 10% relative to a plasmid vector comprising the TEP protein.
Across the independent claim, the core inventive approach is the incorporation of a promoter-controlled TEP protein coding sequence and a MAR element into a transposon vector defined by 5′ and 3′ ITRs, where the combination is stated to increase transgene expression in a mammalian cell by at least 10% relative to a corresponding plasmid vector containing the TEP protein.
Stated Advantages
Increases expression of a transgene encoding a product of interest in a mammalian cell by at least 10% relative to a plasmid vector comprising the TEP protein.
Documented Applications
No documented applications found
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